continuous biohydrogen and biomethane production from whey permeate in a two-stage fermentation...

Continuous Biohydrogen and Biomethane

Production from Whey Permeate in a

Two-Stage Fermentation ProcessM. Kisielewska, I. Wysocka, and M.R. RynkiewiczDepartment of Environment Engineering, University of Warmia and Mazury in Olsztyn, Warszawska St. 117A,10-719 Olsztyn, Poland; [email protected] (for correspondence)

Published online 7 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ep.11890

This study focuses on the exploitation of the two-stagecontinuous fermentation process for biohydrogen and biome-thane production from whey permeate. Mesophilic fermenta-tive biohydrogen production was investigated at a constanthydraulic retention time (HRT) of 24 h and organic loadingrates (OLRs) of 20, 25, 30, and 35 kg COD/m3�d. The hydro-genogenic reactor was successfully operated at OLR of 30 kgCOD/m3�d when the proportion of hydrogen in biogas, volu-metric productionrsquo rate of hydrogen, and hydrogen yieldreached the maximum values of 44.6 6 0.8%, 5.26 6 0.15L H2/d, and 4.19 mol H2/kg CODremoved, respectively. Theeffluent from hydrogenogenic reactor was further digested tobiogas in the second reactor operated at a HRT of 3 d andvariable OLR in the range of 0.9–1.9 kg COD/m3�d. Maxi-mum biogas production rate, volumetric production rate ofmethane and methane yield were 16.90 6 0.6 L/d, 11.95 60.6 L CH4/d, and 0.12 m3 CH4/kg CODadded. The total chemi-cal oxygen demand elimination from whey permeatereached 98% in a two-stage process. VC 2013 American Insti-tute of Chemical Engineers Environ Prog, 33: 1411–1418, 2014

Keywords: hydrogen, renewable energy, anaerobicdigestion

INTRODUCTION

Nowadays, the most widely produced biofuels are bioe-thanol and biogas (methane). In the future energy economy,biohydrogen will play an important role as a clean, CO2-neu-tral energy for use as a substitute for fossil fuels. Today,most hydrogen gas is obtained from fossil fuels which gener-ate greenhouse gases that contribute to global warming [1].Wastewater from food processing industries show greatpotential for economical production of hydrogen [2], buttoday no strategies for industrial-scale productions havebeen developed.

Several strategies for the production of biohydrogen byfermentation in laboratory-scale have been found in the liter-ature: photofermentation [3], dark fermentation [4], and com-bined fermentation, which refers to the two fermentationscombined [5, 6].

In dark fermentation process, complex and problematicorganic wastes can be directly used as a substrate, which sig-nificantly enhances their economic viability. The drawback,

however, in dark fermentation is that the decomposition ofsubstrates is incomplete and organic acids remain, thus limit-ing the biohydrogen yield to <20% of the theoretical maxi-mum value of 12 mol H2/mol glucose [7]. To enhancebioenergy generation and improve total wastewater biode-gradation, a two-stage anaerobic system that dark fermenta-tive hydrogen production is sequentially followed bymethanogenesis has been suggested [5, 8, 9].

Until now the two-stage anaerobic process for biohydro-gen and biomethane production has been operated with var-ious types of organic waste substrates, such as glucose [10],sucrose [11], molasses [12, 13], olive pulp [14], food waste [5,15], and household solid waste [9]. The dairy industry pro-duces highly concentrated, lactose-rich wastewaters and dis-posal of these represent a real problem. There were someexperiences working with cheese whey as the substrate forbiohydrogen and methane production in a two-stage fermen-tation process [8, 16–18]. Because of different results pub-lished, further studies are needed to verify the datasubmitted.

The aim of this study was to investigate the two-stageanaerobic continuous process for the production of biohy-drogen and biomethane from whey permeate.

MATERIALS AND METHODS

Inoculum and WastewaterAnaerobic granular sludge from a full-scale UASB (Upflow

Anaerobic Sludge Blanket) reactor treated fruit juice process-ing wastewater in fruit juice industry, Olsztynek, Poland, wasused as inoculum for biohydrogen and biomethane produc-tion. Prior to inoculation of the hydrogenogenic reactor, thegranular sludge was washed with tap water and then boiledfor 1 h to inactivate hydrogen consuming microorganisms.Finally, the concentration of volatile suspended solids (VSS)seeded into the hydrogenogenic reactor was 30.15 g/L. Nopretreatment of the granular sludge for methane productionwas carried out prior to inoculation of the methanogenicreactor. Initial concentration of inoculum for methane pro-duction was 32.88 g VSS/L. Both reactors were initially ino-culated at a ratio of 35% (by volume).

A solution of dried ultrafiltration (UF) whey permeatefrom the Dairy Plant in Ostrowia Mazowiecka, Poland, wasused as a fermentation substrate. The solution was preparedby dissolving permeate powder in tap water to achieveVC 2013 American Institute of Chemical Engineers

Environmental Progress & Sustainable Energy (Vol.33, No.4) DOI 10.1002/ep December 2014 1411

chemical oxygen demand (COD) concentrations according tothe required OLRs in the hydrogenogenic reactor (Table 1).The general characteristic of wastewater used in this studywas as follow: total sugars (as lactose) 80% v/v, COD/N ratio55 g COD/g N, COD/P ratio 93 g COD/g P.

Hydrogen Production Process Setup and OperationA 5.5 L UASB hydrogenogenic reactor (R1) with 5.0 L

working volume was made of stainless steel and cylindricalin shape (Figure 1). The pH of mixed liquid in R1 was con-trolled automatically at pH 5.7–5.8 (60.05) with 1 M NaOH.The temperature in R1 was maintained at 35�C by insertingthe reactor in the thermostatic chamber. The continuousstartup procedure was used in this experiment, as the fed-bath method was found to be not ideal for startup [19]. Dur-ing continuous startup phase lasting 20 days, the OLR wasmaintained at the level of 20 kg COD/m3�d while the influentflow rate was controlled by a peristaltic pump to maintain aconstant hydraulic retention time (HRT) of 24 h.

During the experiment, four running stages were identi-fied in term of OLR applied with a constant HRT of 24 h(Table 1). The OLR was increased from the initial 20 kgCOD/m3�d to finally 35 kg COD/m3�d in steps of 5 kg COD/m3�d. At each stage the reactors were operated for 17–19times the HRT to reach a steady state at every OLR tested(the steady state was defined as the state that the standarddeviations of COD removal efficiencies were within 5%). TheR1 performance [biogas production and composition, sub-strate removal efficiency (% COD removal) and Volatile FattyAcids (VFAs) concentration and composition] was monitoredevery second day throughout the experimental period.

Methane Production Process Setup and OperationA 5.5 L UASB methanogenic reactor (R2) with 5 L working

volume was made of stainless steel and cylindrical in shape(Figure 1). The pH of mixed liquid in R2 was controlled auto-matically at pH 7.4 (60.05) with 1 M NaOH. The temperaturein R2 was maintained at 35�C by inserting the reactor in thethermostatic chamber. The R2 was fed with the effluent fromR1, which was collected in a 3 L container used as a storagetank (Figure 1). The temperature in the tank was maintained at35�C by placing it in the thermostatic chamber. Overflow efflu-ent flowed out in the top part of the storage tank. The R2 wasoperated at a constant HRT of 3 days. Before R2 was fed withR1 effluent, the diluted UF whey permeate had been used as afeedstock to reach the OLR of 10 kg COD/m3�d and HRT of 2 d(startup procedure). The R2 performance (biogas productionand composition, substrate removal efficiency (% CODremoval) and VFAs concentration and composition) was moni-tored every second day throughout the experimental period.

Analytical MethodsDeterminations of COD and VSS of the anaerobic sludge

were carried out according to the Standard Methods [20]. ThepH in R1 and R2 was measured continuously using the mem-brane electrodes, model ESAgP-301W, Eurosensor, placed inthe liquid phase of each reactor.

Biogas flow rate in R1 and R2 was measured continuouslyusing the two digital gas flow meters XFM17S (AalborgInstruments & Controls).Biogas composition was analyzed byusing a gas chromatograph (GC, 7890A Agilent) equippedwith a thermal conductivity detector (TCD). The GC was fit-ted with the two Hayesep Q columns (80/100 mesh), twomolecular sieve columns (60/80 mesh), and Porapak Q col-umn (80/100) operating at a temperature of 70�C. The tem-perature of the injection and detector ports were 150 and250�C, respectively. Helium and argon were used as the car-rier gases at a flow of 15 mL/min.

The concentrations of VFAs in the liquid samples weremeasured by a gas chromatograph (Br€uker, 450-GC) with aflame ionization detector (FID) and a capillary column typeCP-FFAP CB (25 m 3 0.53 mm). The temperature of theinjection and detector ports was 230�C. The GC oven tem-perature was programmed to increase from 125 to 210�C in8�C min, with a final hold time of 8 min. Helium was thecarrier gas at a flow rate of 1 mL/min with a split ratio of 20.Samples for VFAs determination were acidified by additionof orthophosphoric acid.

RESULTS AND DISCUSSION

Biohydrogen Production in R1The reactor operation was divided into 4 stages according

to increasing in OLR. Table 1 shows the results which are

Table 1. Mean values 6 standard deviation of pH, COD removal efficiency, biogas production rate, hydrogen concentration inbiogas, volumetric hydrogen production rate (VHPR), and hydrogen yield (Y) in R1

Operating conditions

OLR (kg COD/m3�d) 20 25 30 35HRT (h) 24 24 24 24Operating at steady-statepH in R1 5.81 6 0.1 5.80 6 0.1 5.71 6 0.1 5.69 6 0.1COD removal (%) 31.3 6 0.8 32.3 6 0.7 33.1 6 0.5 27.7 6 0.1Biogas (L/d) 6.82 6 0.3 10.28 6 0.4 11.8 6 0.2 10.97 6 0.2Hydrogen (%) 24.6 6 1.3 39.4 6 0.9 44.6 6 0.8 31.2 6 0.9VHPR (L H2/d) 1.67 6 0.07 4.05 6 0.16 5.26 6 0.15 3.42 6 0.06Y (mol H2/kg CODremoved) 2.12 3.97 4.19 2.80Y (ml H2/g CODadded) 16.73 32.42 35.10 19.56

Figure 1. Schematic diagram of the two-stage fermentationsystem.

Environmental Progress & Sustainable Energy (Vol.33, No.4) DOI 10.1002/ep1412 December 2014

calculated as average values under steady state operation,summarizing the effects of OLRs on biohydrogen productionin R1. The increase in OLR from 20 to 30 kg COD/m3�d posi-tively affected both volumetric production rate of hydrogen(VHPR) and hydrogen yield (Figures 2 and 3). The highestVHPR was observed when the OLR was 30 kg COD/m3�dand was 1.05 (60.03) L H2/L�d (5.26 6 0.15 L H2/d; Figure2). Under that condition, the maximum biohydrogen yieldwas observed (4.19 mol H2/kg CODremoved, 35.10 mL H2/gCODadded; Figure 3). A further increase in OLR to 35 kgCOD/m3�d deteriorated the performance of R1, which wasunsteady at the beginning of the stage. During steady state,the production of hydrogen fell to the average value of 0.68(60.01) L H2/L�d (3.42 6 0.06 L H2/d; Figure 2), but it washigher than under the lowest OLR. Generally, up to OLR of35 kg COD/m3�d, the R1 was adapted successfully to thewhey permeate stream, which indicates that the microorgan-isms still had the ability to adapt to the increasing OLR.

The proportion of hydrogen in biogas was also stimulatedby increasing OLR up to 30 kg COD/m3�d. The maximumconcentration of biohydrogen in biogas was 44.6 (60.8)%(Figure 2). During the highest OLR the average hydrogenconcentration was 31.2 (60.9)% which was over 6% greaterthan at the lowest OLR. The production of methane wasunder the detection limit at all the applied OLRs, which indi-cates that methanogenesis was suppressed during the study.This was because the pH was regulated at 5.7–5.8, whichwas unfavorable for methanogens growth but favorable forhydrogen-producing bacteria [13].

In comparison to others researchers, biohydrogen produc-tion from the dairy substrate was different from previously

obtained. Venetsaneas et al. [8] noticed lower production rateof 1.45 L H2/d and nearly half the lower concentration ofhydrogen in biogas (23.8%) using a continuous stirred tankreactor (CSTR) fed with the raw cheese whey (HRT 24 h,OLR 30 g COD/m3�d). Davila-Vazquez et al. [21] using thesame reactor as Venetsaneas et al. [8] but fed with a cheesewhey powder solution reported high VHPR of 28 L H2/L�dwhen the OLR was 142 g lactose/L�d and HRT of 6 h. Anto-nopoulou et al. [17] investigated the potential of hydrogenproduction from raw cheese whey at 35�C in CSTR operatedat HRT of 24 h. The hydrogen production rate was 7.53 LH2/d, while the yield of hydrogen produced was 0.041 m3

H2/kg CODadded. It was higher than obtained in this study(0.035 m3 H2/kg CODadded). A solution of dry whey perme-ate was used by Yang et al. [22]. When the OLR was 14 kgCOD/m3�d and HRT 24 h, the maximum hydrogen yield of2.3 mM/g CODremoved, the maximum hydrogen content inbiogas of 33.2% and VHPR of nearly 1 L H2/d were noticed.Working with an upflow anaerobic packed bed reactor(PBR) fed with cheese whey powder solution rich in lactose(72% w/w), Perna et al. [23] obtained similar VHPR to thisstudy of 1.0 L H2/L�d but significantly low hydrogen contentin biogas of 10% operating with an OLR of 37 kg COD/m3�dand HRT of 24 h.

Methane Production in R2The effluent from R1 collected in a 3 L storage tank

flowed to R2, which was operated at a constant HRT of 3 d.The OLR in R2 depended on COD concentration in R1 efflu-ent and ranged from 4.6 (60.08) to 8.5 (60.10) kg COD/

Figure 2. OLR-dependent profile of volumetric hydrogen production rate (VHPR) and hydrogen concentration in biogas in R1.

Figure 3. OLR-dependent profile of hydrogen yield (Y) and COD removal efficiency in R1.


m3�d (Table 2). Table 2 shows the results which are calcu-lated as average values under steady state operation, summa-rizing the effects of OLRs on methane production in R2. TheR2 was operated efficiently throughout the study, but thebest results were obtained when the OLR in R1 was 25 and30 kg COD/m3�d and when the R1 reached the best perform-ance. The highest biogas production rates were 16.90 (60.6)L/d and 16.81 (60.5) L/d while volumetric production rate ofmethane (VMPRs) were 10.54 6 0.6 L CH4/d (2.12 6 0.1 LCH4/L�d) and 11.85 6 0.6 L CH4/d (2.39 6 0.1 L CH4/L�d),respectively (Figure 4). Methane yield was unchanged duringthe study (0.12 m3 CH4/kg CODadded), but when the OLR inR1 was the highest, the methane yield was down to 0.05 m3

CH4/kg CODadded (Figure 5). The composition of the effluentfrom R1 in the last stage of experiment could create theunfavorable conditions for methanogenic bacteria because of

high amount of acetic and butyric acids as well as higheramounts of propionic acid. The acetic acid can be directlyused to give methane by acetoclastic methanogenesis whilethe butyric acid is first broken down to acetic via the syntro-phic hydrogen production route, with the hydrogen subse-quently being combined with carbon dioxide to formmethane by autotrophic methanogens [18]. The propionicacid can lead to lower efficiency of methanogenic phase fol-lowed the hydrogenogenic phase [24].

Methane content in biogas evolved from R2 ranged from49.03 to 72.36% during the experimental period (Figure 4).The highest average concentration (70.47% 62.1) was obtainwhen the OLR in R1 was 30 kg COD/m3�d. Hydrogen wasnot detected in biogas.

Antonopoulou et al. [17] investigated the two-stage anaer-obic process for biohydrogen and methane production from

Figure 4. OLR-dependent profile of biogas production rate, volumetric methane production rate (VMPR), and methane con-centration in biogas in R2.

Figure 5. OLR-dependent profile of methane yield (Y) in R2.

Table 2. Mean values 6 standard deviation of pH, COD removal efficiency, biogas production rate, methane concentration inbiogas, volumetric methane production rate (VMPR), and methane yield (Y) in R2

Operating conditions

OLR (kg COD/m3�d) 4.6 6 0.08 5.9 6 0.48 6.9 6 0.26 8.5 6 0.10HRT (d) 3 3 3 3Operating at steady-statepH in R2 7.42 6 0.05 7.39 6 0.02 7.35 6 0.05 7.27 6 0.05COD removal (%) 97.1 6 0.2 96.7 6 0.3 97.0 6 0.1 90.6 6 1.2Biogas (L/d) 14.28 6 1.0 16.90 6 0.6 16.81 6 0.5 13.67 6 0.3Methane (%) 58.18 6 1.1 62.36 6 1.5 70.47 6 2.1 54.63 6 1.0VMPR (L CH4/d) 8.32 6 0.7 10.54 6 0.6 11.85 6 0.6 7.47 6 0.3Yield (m3 CH4/kg CODadded) 0.12 0.12 0.12 0.05


raw cheese whey. They achieved higher biogas and methaneproduction rate of 105.9 and 75.6 L/d, respectively in a peri-odic anaerobic baffled reactor (PABR) operated at HRT of 4.4d. Using cheese whey as a substrate for biohydrogen andbiomethane production, Venetsaneas et al. [8] obtainedapproximately 1 L CH4/d (0.4 L CH4/L�d) in CSTR-type reac-tor fed with the effluent from hydrogenogenic reactor andoperated at HRT of 20 d.

Comparison with Other Two-Stage Anaerobic DigestionMany researches have studied the two-stage anaerobic

digestion processes in the two separate reactors and varioustypes of substrate have been used (Table 3) [5, 8, 9, 12–14,17]. The best results were achieved by Kongjan et al. [13]used desugared molasses as a substrate. During steady stateconditions, the average hydrogen and methane concentra-tions in biogas were 45 and 75%, respectively. Such goodresults in VHPR and VMPR were probably due to tempera-ture conditions (55�C). Fermentation at higher temperatureyielded higher biohydrogen formation rate due to elimina-tion of hydrogen consuming bacteria at high temperatures,which were active in mesophilic conditions, reducing biogassolubility and lowering H2 partial pressure in the head space[5, 25]. According to Vindis et al. [26] thermophilic anaerobicdegradation is up to 8 times faster and 4 times more intense,has higher VSS removal efficiency and yields more biogasthan mesophilic degradation. At mesophilic conditions maxi-mum VHPR was 2.1 or 2.9 L H2/L�d from cheese whey [8, 17]and 2.8 L H2/L�d from molasses [12] (Table 3). The VMPRswere lower than VHPR and ranged from 0.4 to 5.0 L CH4/L�d(Table 3). At this study the VMPR was higher than VHPR and

achieved 2.37 L CH4/L�d and 1.05 L H2/L�d, respectively. Sim-ilar trend was obtained by Liu et al. [9] and Koutrouli et al.[14].

Substrate DegradationUtilization of whey permeate for biohydrogen and meth-

ane production was evaluated in terms of substrate degrada-tion rate (kg CODremoved=m

3reactor � d) and substrate removal

efficiency (% COD removal; Figures 3 and 6).Maximum substrate removal efficiency in R1 of 33.1

(60.5)% was observed when the OLR was 30 kg COD/m3�d,followed by 32.3 (60.7)% at OLR of 25 kg COD/m3�d and31.3 (60.8)% at the lowest OLR. Substrate degradation ratewas found to increase with loading conditions from 20 to 30kg COD/m3�d (Figure 3). The average values observed at asteady state conditions was 6.26 (60.15) kg CODremoved/m

3�dat OLR of 20 kg COD/m3�d, followed by 8.08 (60.18) kgCODremoved/m

3�d at OLR of 25 kg COD/m3�d and 9.94(60.15) kg CODremoved/m

3�d at OLR of 30 kg COD/m3�d(Figure 6). A further increase in OLR to 35 kg COD/m3�dresulted in a decrease of substrate degradation to 9.68(60.04) kg CODremoved/m

3�d. On the contrary to substratedegradation, the substrate removal efficiency under the high-est OLR was the lowest [27.7 (60.1)% at steady stateconditions].

It was similar to results achieved by Mohanakrishna et al.[27]. They reported 9.19 kg CODremoved/m

3 (34.75%) whenthe OLR was 26.44 kg COD/m3�d and 9.63 kg CODremoved/m3 (27.30%) when the OLR was 35.25 kg COD/m3�d duringbiohydrogen production from vegetable based solid waste(HRT 24 h). Perna et al. [23] achieved lower COD removal

Table 3. Previous researches about two-stage anaerobic continuous process for biohydrogen and biomethane from differentsubstrates (N.C. – non control)

Operation parameters

SubstrateReactor typefor H2/CH4

Temp. forH2/CH4 (�C)

pH forH2/CH4

HRT forH2/CH4 (h/d)

Max. VHPR(L/L/d)

Max. VMPR(L/L/d) Ref.

Desugared molesses UASB/UASB 55/55 N.C. 16/3 5.6 3.38 [13]Cheese whey CSTR/CSTR 35/35 N.C. 24/20 2.9 0.4 [8]Molasses PBR/PBR 35/35 5.5/7.0 6/4 2.8 1.94 [12]Potato CSTR/CSTR 55/55 5.5/N.C. 48/10 2.1 1.2 [5]Cheese whey CSTR/PABR 35/35 N.C. 24/4.4 2.1 5.0 [17]Household solid

wasteCSTR/CSTR 37/37 N.C. 48/15 1.6 2.5 [9]

Whey permeate UASB/UASB 35/35 5.71/7.35 24/3 1.05 2.37 This studyOlive pulp CSTR/CSTR 35/35 5.0/7.62 30/20 0.26 0.64 [14]

Figure 6. OLR-dependent profile of total COD removal efficiency, COD removal efficiency in R2 and substrate degradation inR1 and R2.


values ranged between 14 and 32% when the hydrogeno-genic reactor was operated with the increasing OLRs from 25to 37 kg COD/m3�d and diluted cheese whey powder wasused as a substrate (HRT 24 h); 17.7 (63.1)% COD removalefficiency was reported by Park et al. [12] during continuousproduction of hydrogen from molasses (1082.2 g COD/L) inthe two-stage anaerobic digestion process (HRT 24 h).

In the two-stage anaerobic process, the aim of the first-stage degradation is to produce biohydrogen as well as VFAsfor further degradation in the methanogenic reactor. In thesecond stage (R2), maximum substrate degradation (averageof 23.07 kg CODremoved/m

3�d) was achieved at the highestOLR in R2 of about 8.5 kg COD/m3�d, followed by 19.99 kgCODrmoved/m

3�d at OLR in R2 of about 6.9 kg COD/m3�d,17.21 kg CODremoved/m

3�d at OLR in R2 of about 5.9 kgCOD/m3�d and on average 13.42 kg CODremoved/m

3�d at thelowest OLR of 4.6 kg COD/m3�d (Figure 6). Although thesubstrate degradation increased along with the OLRs, thesubstrate removal efficiency was found to decrease fromover 97% to 90.6 (61.2)% (Figure 6).

Using cheese whey as a substrate in a two-stage continu-ous process, Venetsaneas et al. [8] also reported high CODremoval efficiency of 95.3% in methanogenic reactor butwith a longer HRT of 20 d and OLR about 2.5 kg COD/m3�d.Other researchers reported 94% COD reduction in the meth-anogenic stage from raw cheese whey as a substrate [17].Cota-Navarro et al. [16] obtained COD removals above 90%in methane producing reactor at OLR and HRT of 20 g COD/L�d and 6 h, respectively in a two-stage cheese whey fermen-tation system.

The total substrate removal efficiency in R1 and R2 washigh in all stages and was above 92% throughout the study(Figure 6). It was higher than reported by Liu et al. [9], whohave noted only 86% substrate removal efficiency for overalltwo-stage process from household solid waste. Park et al.[12] obtained the overall COD removal efficiency of 79.8%during a two-stage fermentation of molasses.

Fermentation ProductsBiphasic production of hydrogen and methane from

whey permeate resulted in VFAs production summarized inFigures 7 and 8. The predominant fermentation productswere n-butyric and acetic acids (HAc), which was expectedon the basis of the work of other researchers [8, 17]. Theconcentration of butyric acid (HBu) in R1 effluent was in therange of 2.30–5.06 g/L when the OLRs were 20–30 kg COD/m3�d (Figure 7). Under the highest OLR of 35 kg COD/m3�dit increased and amounted to an average value of 12.40 g/L.The concentration of isobutyric acid (i-HBu) remained at alow level below 1.59 g/L while acetic acid was in the rangeof 3.61–8.89 g/L during all stages of experiment (Figure 7).Propionic acid (HPr) was only detected in R1 effluent underthe OLR of 30 and 35 kg COD/m3�d, but it stayed at lowlevel averaged 0.47 and 0.87 g/L, respectively (Figure 7).Valeric and isovaleric acids were not detected at all. In thestudy of Venetsaneas et al. [8], butyric acid (14.50 g/L) andacetic acid (9.20 g/L) were the dominant metabolic productsin the effluent from the hydrogenogenic reactor using cheesewhey as a substrate (35�C, HRT 24 h, OLR 30 g COD/d).

Figure 7. OLR-dependent profile of VFAs in R1.

Figure 8. OLR-dependent profile of VFAs in R2.


Methanogenic reactor provided high removals of bothacetic and n-butyric acids over all runs (Figure 8). In the firstthree stages of experiment, the total VFAs concentration wasin the range of 0.10–0.47 g/L and acetic acid was the mainVFA species averaged 79.02% of the total VFAs. It was due tothe anaerobic digestion of other VFAs from R1 effluent intoacetate that was further digested into methane by acetotro-phic methanogens [12]. Moreover, significantly lower VFAs inR2 effluent means that methanogens worked with a satisfac-tory yield. The average reduction of VFAs in R2 was 97.8% 61.2. Under the highest OLR in R2 of 8.5 kg COD/m3�d, theconcentration of VFAs in the effluent from methanogenicreactor increased to the average level of 1.86 g/L implyingVFAs accumulation and deterioration of the reactor perform-ances (Figure 8). The average reduction of VFAs in R2 underthe steady state conditions decreased to 88.4% 6 0.6. Besidesacetic acid, butyric acid (averaged 7.15% of the total VFAs)as well as propionic acid (averaged 34.03% of the total VFAs)were detected in the effluent. Similar trends were alsoobserved by Kongjan et al. [13]. Increasing OLR resulted inpropionic and butyric acid production in methanogenicstage.

CONCLUSIONS

The performance of two-stage continuous fermentationprocess with combined hydrogenogenic and methanogenicUASB reactors was evaluated on laboratory scale.In theexperimental optimization of the biohydrogen production,the process was stable up to 35 kg COD/m3�d, but the beastresults were achieved when the OLR was in the range of 25–30 kg COD/m3�d. The maximum biohydrogen productionwas 5.26 6 0.15 L H2/d, while the hydrogen yield understeady state operation reached 4.19 mol H2/kg CODremoved

(35.10 mL H2/g CODadded). Methane production using hydro-gen effluent as a substrate occurred with a satisfactory yieldin all stages. In the methanogenic process, the methane yieldwas stable when the OLR in the hydrogenogenic reactor wasin the range of 20–30 kg COD/m3�d and was 0.12 m3 CH4/kgCODadded. The maximum methane production was 11.95 60.6 L CH4/d. Under the best conditions, the overall CODremoval efficiency of the two-stage process was determinedto be 98%.

These results indicate that whey permeate could be effi-ciently used for hydrogen and methane production and thetwo-stage fermentation process could be operated success-fully under these experimental conditions. Permeate streams(whey and milk UF permeates) are high strength wastewaterscharacterized by high COD concentrations [8, 16]. Therefore,further studies should be done to evaluate the possibility ofoperation with much higher OLRs in the hydrogenogenicand methanogenic reactors.

ACKNOWLEDGMENTS

This work was supported by the National Science Centre,Poland [N N 523 555138].

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